This invention relates to density gradient centrifugation separations and, more particularly, to reorienting the density gradient in vertical tubes to enhance the speed of separation and facilitate recovery of the separation zones.
The field of centrifuging is a relatively old field. It is based upon the use of centrifugal force to separate particles. Such force causes the particles to move outwardly from the rotational center of the rotor towards the periphery. This is called sedimentation. The sedimentation rate is dependent upon several factors. These factors include rotational speed, the density and viscosity of the medium in which the particles are suspended, the density of the particle, and the size and shape of the particle.
Utilizing these various criteria, the particles are separated in space by the differing distances they traverse along the centrifugal force vector. The degree of separation along this force vector, often termed a separation gradient, determines the degree of resolution with which particles may be separated.
In one type of separation, known as density gradient separation, a column of liquid in which the density of the liquid varies in a known way from one end of the column to the other, is used. Thus when the particle under the influence of centrifugal force reaches the point of its isopycnic density, i.e., the density of the surrounding liquid, it will cease to migrate along the force vector in either direction.
Swinging bucket centrifuges typically are used for this purpose. A disadvantage of this type of separation is that the required separation times can be extreme, even when ultra centrifuges are used, because of the relatively long path length the particles must travel to be separated from one another. While a disadvantage in separation time, the container length is a decided advantage in recovery of the separation zones or bands. The long column provides for a relatively wide separation of the bands of particles which is a decided advantage. It would be desirable to achieve shorter separation times and yet retain the advantages of long path length during recovery.
In addition to swinging bucket rotors, zonal centrifuge rotors have been devised for density gradient separation work. One such is described in U.S. Pat. No. 3,243,105 issued Mar. 29, 1966 to Norman G. Anderson. Anderson provides a bowl type rotor in which a density gradient is established. The problems encountered with the Anderson approach are many and are based to a large extent upon the fact that the spin axis passes directly through the bowl. This means that portions of the sample must go through relatively large area changes with attendant, excessive shearing. This excessive shearing tends to disturb the separation and therefore decreases the resolution and purity of separation.
An improvement over Anderson is described in U.S. Pat. No. 3,708,111 issued Jan. 2, 1973 to Phillip Sheeler et al. There is described in Sheeler et al a reorienting gradient zonal rotor in which the rotor is a cylindrical chamber divided into a plurality of sector-shaped compartments. The floor or ceiling of the chamber defines a U-shaped annular groove to increase the path length and hence increases the separation of the bands as they are recovered. Unfortunately the separation path length is relatively long with the attendant separation time disadvantages.
Accordingly, it is the object of this invention to obviate many of the disadvantages of the prior art methods of centrifuge gradient separations.
Another object of this invention is to provide an improved method of density gradient centrifugation.